A “Hands-on Workshop on FISH and its application in Pathology and Clinical Medicine” was organized by MetaSystems and Department of Pathology, Himalayan Institute of Medical Sciences (HIMS) to demonstrate the applications of FISH in hematological malignancies and solid tumors.
Sample Digitization, Analysis, and Sharing
Combine smart, all-round scanning solutions with outstanding flexibility.
With the slide scanning platform Metafer MetaSystems also offers an extensive toolbox to digitize, analyze, and share larger microscopic samples such as tissue sections. Using sophisticated algorithms, dedicated software for the composition of larger images (VSlide) generates high-quality digital copies of a slide. Such images can provide focus stacks, metadata such as information on the location of cells, and sub-images from different origins. In a free viewer (VSViewer) users can create their own annotations, generate snapshots from the main image, display different color channels separately on the same screen, and apply contrast enhancement algorithms. Tools within Metafer allow users to interpret tissue micro-array cores, or to match consecutive tissue sections. Neon integrates all workflow components and keeps track of the process.
VSlide combines the advantages of a motorized microscope with modern, high-quality imaging automation. The system is not restricted with respect to magnifications or contrasting modes. It can take advantage of any microscope feature, and it can combine different methods to a smart scanning workflow. Even Z-stacks are automatically acquired and stored in the image files so that the users can virtually 'focus' through the digitized sample.
Imaging with Metafer is very easy. The system automatically reads and interprets bar code labels, and configures the scan according to the information in the code. Pre-scans facilitate tissue detection in bright field, transmitted light, dark field, or any other contrasting method. A high resolution scan can be added either automatically, based on the spatial data obtained in the pre-scan, or on selected regions interactively determined by the researcher.
Metafer’s virtual slide image is generated automatically in the background while the system scans. Parameter sets define the employed color channels and the file format of the final image. Since the source image files are kept on the system, new virtual slides with other parameters can be re-created anytime. Metafer users can choose from a variety of output file formats, including the comprehensive, proprietary VSI format. VSI images contain all relevant image information including color channel details, focus stacks, and annotations. In the local network, VSI images are read with the versatile viewing software (VSViewer).
Once a VSI file is created, all the spatial information of the image tiles originally acquired remains accessible. This allows for precise relocation of any sample region under the microscope. Also subsequent acquisitions of selected regions with different techniques are possible. This unique feature allows for setting up an unlimited number of smart workflows. Regions of interest (e.g., tumor regions in an H&E section) can be easily marked in the viewing software. Once a list of target regions is available, they can be re-scanned anytime with other magnifications, contrasting methods, or other scanning parameters. The selection of regions can even be done remotely, and the tissue matching tool of Metafer facilitates the transfer of the regions to another sample, e.g., for imaging the same region in subsequent tissue sections.
The free viewing software for digital samples created by Metafer (VSViewer) can be installed on any Windows computer with network access to the image data. The simplified, modern user interface offers all customary viewing tools offered by handy toolbars which can be hidden, minimized, snapped to the window corner, or freely arranged on the desktop. Panning and zooming are done with the mouse and the mouse wheel.
The viewer has many options for displaying image metadata. For example, current Metafer coordinates can be visualized as grids on the image. Also the location of the fields of view (FOV), that are, the single original camera images, can be shown as an overlay to the image. The navigation bar at the bottom of the screen always provides information on the sample location currently shown. This includes the current magnification, the zoom factor, the focus level (if the current image contains focus stacks), and the Metafer coordinate of the window center. Quick access buttons allow for selecting a certain magnification, viewing the complete image, and accessing focus levels. Of course there is also a navigator toolbar showing a thumbnail of the whole image and an indicator of the current section.
Two annotation toolbars make it easy to highlight regions of interest. The ‘Scan Regions’ toolbar is used to prepare the next scan of a selected part of the sample. A quick selection of the target region generates an annotation file which is directly read by Metafer. The ‘Annotations’ toolbar then offers a comprehensive range of tools to annotate and highlight items in the image. If the current image originates from an object search in Metafer, it is even possible to visualize the detected objects within their original vicinity and to group them by any obtained analysis result.
The image window can be split into up to four coupled sections. With the ‘Channels’ toolbar it is then possible to assign different display parameters to each sub-window. Hence, it is possible to visualize the same part of the image with different color channels, overlays, and contrasts. Furthermore, it is possible to open two different images, e.g., from subsequent tissue sections, in two viewer software instances which can also be coupled for panning and zooming.
Any view can be exported into standard image file formats (TIFF, JPG, BMP, GIF) as a snapshot. Snapshots may contain additional data such as a magnification ruler, annotations, FOV indications, and even a comment. Additionally, extracts of the image can be interactively defined or by their coordinates, and they can then be saved as a separate image.
MetaSystems has designed a system of hybridization and imaging tools to be integrated in the routine workflow of the pathology lab. Core of this system is the combination of dedicated DNA probes for tissue sections with the innovative platform Metafer, a flexible and robust slide scanning system with many options.
MetaSystems Probes‘ XL range of locus-specific fluorescence-in-situ DNA probes for tissue FISH gene amplifications, deletions, or translocations involved in solid tumors (e.g., ALK, EGFR, HER2/neu, MYC, and many more). Intense signals aid with the interpretation on tissue sections. The Metafer scanning platform automatically generates virtual slides in brightfield and fluorescence, acquires high resolution FISH images and provides an automatic score. Equipped with an automated bar code reader and the robust SlideFeeder x80 for up to 800 samples, it can be operated in a 24/7 mode.
- As initial step a digital brightfield, e.g. H&E slide is generated by Metafer. The pathologist calls up the digital slide and selects on screen the tumor region on the virtual slide that needs to be FISH-scored.
- Next, the FISH slide (from a subsequent section of the same block) is being scanned at low magnification to generate an overview. Displayed side by side to the marked digital H&E image the tumor region can easily be transferred to the FISH slide.
- Metafer now has all the information to start automatic image acquisition of the FISH slide at higher magnification. Cell nuclei that are isolated or slightly connected will be separated automatically and spot-counted. Manual tools for segmentation help to separate touching nuclei for immediate automatic scoring until the preset number of cells to be analyzed has been reached. The software module can be easily set up to match the individual analysis standards. For instance, it is possible to define a minimum number of cells to be analyzed, and also to define a number of independent readers.
More on signal analysis in tissue sections can be seen here.
- For final review the system presents a full synopsis to the pathologist, comprising the cell gallery with the scoring results, the virtual DAPI slide showing the positions of analyzed cells, and the corresponding H&E virtual slide. Every cell can be traced back to the tissue section to confirm its location within the preselected tumor region.
- Final results can either be exported as raw data, e.g. for subsequently being processed by external software, or can be summarized in comprehensive, user-adaptable reports.
The laborious nature of procedures, shortages of sample material, and limitations in the cost of reagents for diagnostics in routine clinical pathology has raised the interest in high throughput solutions. The tissue micro-array (TMA) technique addresses these issues by obtaining small tissue cores from regions of interest in paraffin embedded tissues, assembling them in an array-like fashion, and mounting them to a standard glass slide for microscopy. Each of these cores then represents an independent analysis case.
Due to the special layout of samples automation of TMA has to be done based on the following considerations:
- Analysis should take the general layout of the TMA into account,
- It should be possible to correct contortions, rotations, or other spatial aberrations generated during slide preparation,
- Each TMA core should be clearly identifiable by a label,
- Analysis results should be separable so that data can be assigned to the single core.
With the unique combination of Metafer's scanning capabilities, the integrated tool for TMA analysis, and the sample digitization software, MetaSystems offers a complete package for precise and effortless automated TMA analyses. Once a tissue microarray (TMA) is created, its layout can be expressed with the number of cores per row, the number of rows in the array, the diameter of the cores, and the distance of cores. In the integrated Metafer TMA tool these values can be used to generate a TMA map file that is used as basis for the analysis. Additionally each core can receive a unique identifier.
A fast pre-scan at low magnification is used to generate a map of the current slide. The result of the pre-scan is displayed side by side with the TMA map, and with a flexible grid the actual image can be matched to the map in order to correct any spatial aberrations. Missing or invalid cores can be marked in this interface. On confirmation the TMA tool converts the data into a position list, which contains the exact positions of each core, and also the core identifiers. The system is now ready to relocate each core and acquire and analyze the core images. If the image stitching software is available, core images are automatically composed in the background and an overview image of the whole TMA is generated.
Our sister company MetaSystems Probes introduces a probe panel covering a subset of the most relevant chromosomal loci associated with the development of sarcomas. All probes are intended for hybridization on methanol/acetic-acid fixed cells and formalin-fixed, paraffin-embedded (FFPE) tissue sections. In combination with the optimized TissueFISH Pretreatment Kit, this panel is the perfect supplement to any MetaSystems device for tissue analysis.
On the web site of MetaSystems Probes you can find a list of all probes available for tissue FISH.